Amelioration of intestinal fibrosis and treatment of Crohn&#39;s disease

ABSTRACT

Methods of treating patients with inflammatory bowel disease, intestinal fibrosis, or Crohn&#39;s disease involve administering a therapeutic amount of CARD-024 or related compound.

CROSS REFERENCE TO EARLIER APPLICATIONS

This application claims the benefit of U.S. provisional application Ser.No. 61/746,969 filed Dec. 28, 2013.

GOVERNMENT SUPPORT

This invention was made with government support under DK080172 andHL074894 awarded by the National Institutes of Health. The Governmenthas certain rights in the invention.

INTRODUCTION

Crohn's disease (CD) is a chronic, progressive intestinal disordercharacterized by cycles of intestinal inflammation and mucosal healing.Despite the advent of powerful anti-inflammatory therapies, 70% ofpatients ultimately develop fibrostenotic disease for which there are noeffective pharmacologic therapies (Andres and Friedman, 1999; Cosnes etal., 2002; Loftus, 2004). Patients with CD have an increased prevalenceof vitamin D deficiency, but it is not clear whether vitamin Ddeficiency is a contributing factor to fibrosis, or merely a diseaseconsequence (Harries et al., 1985; Siffledeen et al., 2003). In otherorgan systems, including kidney, liver, lung, skin and heart, vitamin Ddeficiency is associated with fibrosis (Li et al., 2005; Rahman et al.,2007; Ramirez et al., 2010; Tan et al., 2006; Weishaar et al., 1990;Zhang et al., 2011).

Vitamin D analogs have been shown to reduce fibrosis in cell culture andanimal models of cardiac, kidney, and renal fibrosis (Li et al., 2005;Mancuso et al., 2008; Tan et al., 2006; Zhang et al., 2010). In CD,activated subepithelial myofibroblasts are the major contributor tointestinal fibrosis (Powell et al., 1999; Tomasek et al., 2002). Incolonic myofibroblasts, TGFβ induces differentiation and a pro-fibroticphenotype, characterized by stress fiber formation (Brenmoehl et al.,2009; Simmons et al., 2002) and induction of “-smooth muscle actinprotein expression. In other myofibroblast lineages, including lung,heart, and kidney, extracellular matrix (ECM) stiffness alone stimulatesa pro-fibrotic phenotype (Arora et al., 1999; Liu et al., 2010; Olsen etal., 2011), however the effect of matrix stiffness upon colonicmyofibroblasts is unknown.

Though calcitriol (vitamin D) and vitamin D analogs have been clinicallyused for several diseases, including hyperparathyroidism, toxichypercalcemic effects have been reported, underscoring the need for avitamin D analog with minimal hypercalcemic effects.

SUMMARY

CARD-024 attenuated the pro-fibrotic response of colonic myofibroblaststo high matrix stiffness, suggesting that CARD-024 and analogouscompounds can ameliorate intestinal fibrosis. Hence, a therapy fortreating intestinal fibrosis, inflammatory bowel disease, or Crohn'sdisease involves administering to a subject diagnosed with the conditionor disease a composition containing a therapeutically effective amountof a compound of Formula (I)

wherein Et is ethyl, wherein the dashed line indicates a single bond ora double bond in the E configuration between carbon 22 and carbon 23,and wherein the configuration at carbon 24 to which Et is attached is inthe R configuration or the S configuration.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1—Effect of CARD-024 on TGFβ stimulated colonic myofibroblasts. (A)Representative Western blot of αSMA protein expression in Ccd-18co cellsstimulated with increasing doses of TGFβ (0.1-2 ng/ml). (B)Representative Western blot illustrating αSMA protein expression inresponse to treatment with TGFβ and increasing amounts of CARD-024(10-1000 nM) compared to TGFβ treated or untreated cells. In (A) and (B)GAPDH expression is used as a protein loading control. (C)Quantification of αSMA protein expression as normalized to GAPDH inCcd-18co cells treated with TGFβ and 10-1000 nM CARD-024. Results arefrom three independent experiments. (* pb0.05, *** pb0.001).

FIG. 2—Effect of increasing matrix stiffness on myofibroblast phenotype.Brightfield photomicrographs of colonic myofibroblasts cultured onsubstrates of increasing matrix stiffness of 0.01% (2.6 kPa), 0.02% (4.3kPa), 0.08% (15.6 kPa), and 0.16% (28.1 kPa). Myofibroblasts cultured ona standard plastic substrate are shown for comparison.

FIG. 3—Effect of CARD-024 and matrix substrate stiffness on cellmorphology and growth in colonic myofibroblasts. (A) Phase-contrastmicrographs of untreated myofibroblasts (upper panel) on low and highmatrix stiffness compared to myofibroblasts treated with 1000 nMCARD-024 at low and high stiffness (lower panel). 100× magnification.(B) Cell length of untreated (white bars) compared to CARD-024 treatedcells (dark bars) on low or high stiffness matrices. (C) Cell densitycounts of untreated myofibroblasts (white bars) compared to CARD-024(dark bars) on low or high stiffness matrices. The horizontal referenceline denotes baseline (untreated) expression. Asterisks denotestatistically significant comparisons between untreated/low stiffnessand other experimental groups. ** p<0.01, *** p<0.001 (D) Confocalmicrographs of myofibroblasts on low or high matrix substrates treatedwith 1000 nM CARD-024 compared to untreated cells using Alexa555-conjugated vinculin (red) and phalloidin (green). Nuclei werevisualized by DAPI (blue). The scale bar represents 100 μm.

FIG. 4—Effect of CARD-024 and substrate stiffness ECM protein and geneexpression. (A) Representative Western of αSMA protein expression inuntreated or CARD-024 treated colonic myofibroblasts on low or highstiffness substrates. (B) Representative Western of pFAK proteinexpression in untreated or CARD-024 treated colonic myofibroblasts onlow or high stiffness substrates. In (A) and (B) GAPDH proteinexpression is used as a control for protein loading. (C-H) QRT-PCRexpression of fibrogenic (MLCK (C), ET-1 (D) or COX-2 pathway (PTGS2(E), IL-1β (F)) genes in colonic myofibroblasts cultured on low or highstiffness substrates treated with CARD-024 (dark bars) compared tountreated cells (white bars). Asterisks denote comparisons betweenuntreated/low stiffness and other groups. The horizontal reference linedenotes baseline (untreated/low stiffness) expression. Pairwisecomparisons between groups are denoted with brackets. * p<0.05, **p<0.01, *** p<0.001.

FIG. 5 gives structures of active compounds.

DESCRIPTION

The following abbreviations are used: CD is Crohn's disease; ECM isextracellular matrix; TGFβ is transforming growth factor beta; and αSMAis alpha-smooth muscle actin. Formula (I) and Formula (II) are annotatedwith IUPAC numbering on some of the carbon atoms for clarity and easyreference. “Mcg” or “mcg” is used as an abbreviation for micrograms.

In one embodiment, a method of treating a subject to relieve thesymptoms of inflammatory bowel disease involves administering to thesubject a therapeutically effective dose of a compound of Formula (I)

In Formula (I), wherein Et is ethyl, the dashed line indicates a singlebond or a double bond in the E configuration between carbon 22 andcarbon 23, and the configuration at carbon 24, to which Et is attached,is in the R configuration or the S configuration. Mixtures of compoundsof Formula (I) can also be administered. In a particular embodiment, thecompound of Formula (I) includes CARD-024.

In various embodiments, the method includes administering to the subjecta composition containing a compound of Formula (I) and apharmaceutically acceptable carrier. Advantageously, the composition isformulated as a tablet or a capsule, in non-limiting examples. In theseand other embodiments, the method can be used to treat a subjectdiagnosed with or otherwise exhibiting the symptoms of Crohn's disease.

In another embodiment, a method of ameliorating intestinal fibrosis in asubject involves administering to the subject a composition comprising acompound of Formula (I). In a particular embodiment, the compound ofFormula (I) has a single bond between carbon 22 and carbon 23, whereinin addition the carbon at position 24 is in S configuration, the Rconfiguration, or a mixture of R and S. In another particularembodiment, the compound of Formula (I) has a double bond in the Econfiguration between carbon 22 and carbon 23, again with the asymmetriccarbon 24 being in the R configuration, the S configuration, or in acombination of R and S. In non-limiting examples, the subject to whomtreatment is administered is diagnosed with inflammatory bowel diseaseor with Crohn's disease. In various embodiments, the subject is a humansubject.

In yet another embodiment, method for treating a subject diagnosed withCrohn's disease includes administering to the subject a compositioncomprising a therapeutically effective amount of a compositioncontaining a compound according to Formula (I) or Formula (II) and apharmaceutically acceptable carrier

In a particular embodiment, the composition comprises CARD-024. In otherembodiments, the asymmetric carbon 24 is in the R,S configuration, inthe R configuration, or is in the S configuration. In variousembodiments, suitable doses to be administered in the methods hereinrange from 5 mcg to 500 mcg of the compound. In embodiments, 1 to 4doses per day of the composition are administered to the subject. In anexemplary embodiment, the subject is treated by administering thecompound of Formula (I) in a delayed release composition containing 20to 1000 mcg of the compound, for example once a day.

Compounds of Formula (I) have been found to be effective in a modelsystem, where they attenuate the pro-fibrotic response to TGFβ orsubstrate stiffness in colonic myofibroblasts. As such they arecandidates for treating certain symptoms related to diseases,conditions, or syndrome involving a pro-fibrotic response.

Intestinal fibrosis is one of the major complications of Crohn's disease(CD) for which there are no effective pharmacological therapies. VitaminD deficiency is common in CD, though it is not known whether this is acontributing factor to fibrosis, or simply a consequence of the diseaseitself. In CD, fibrosis is mediated mainly by activated intestinalmyofibroblasts during remodelling of extracellular matrix in response towound healing.

In the present teachings, we investigated the effects of CARD-024, acompound with minimal hypercalcemic effects, on the pro-fibroticresponse of intestinal myofibroblasts to two fibrogenic stimuli: TGFβstimulation and culture on a physiologically stiff matrix comparable toa Crohn's disease stricture. TGF-β1 stimulated a fibrogenic phenotype inCcd-18co colonic myofibroblasts, characterized by an increase in actinstress fibers and mature focal adhesions, increased αSMA proteinexpression, and induction of fibrogenic genes including col1A1, Fn1,MLCK, and ET-1. CARD-024 repressed αSMA protein expression in adose-dependent manner but had minimal impact on fibrogenic geneexpression.

Culture of colonic myofibroblasts on physiological high stiffnesssubstrates induced morphological changes with increased actin stressfibers and focal adhesion staining, induction of αSMA proteinexpression, FAK phosphorylation, induction of fibrogenic genes, andrepression of COX-2 and IL-1β. CARD-024 treatment did repress thestiffness-induced morphological features including stellate cellmorphology and the maturation of focal adhesions. While CARD-024 did notrepress pro-fibrotic genes col1A1 and Fn-1, CARD-024 repressed thestiffness-mediated induction of αSMA protein expression, FAKphosphorylation, and MLCK and ET-1 gene expression. In addition,CARD-024 partially stimulated members of the COX-2/IL-1β inflammatorypathway.

CARD-024 attenuated the pro-fibrotic response of colonic myofibroblaststo either TGFβ stimulation or high matrix stiffness, suggesting thatCARD-024 and structural analogs can ameliorate intestinal fibrosis.

Although the invention is not limited to any theory, it is consideredthat 24 ethyl derivatives would have similar biological activity basedupon the CARD-024 results with CARD-024. The 24-ethyl substituent ispostulated to prevent the normal hepatic 25-hydroxylation, making the24-ethyl derivatives more hydrophobic than a normal 25 hydroxylated Danalog. This would lead to different localization (e.g., membrane vs.cytosol) and different biological activity. The 24-ethyl substituent isthe component uniting all the structures herein described for Crohn'sand other therapies.

Compounds of Formula (I) are shown in FIG. 5.

Compound Ia is CARD-024, named as(1S,3R)-(5Z,7E)-9,10-Secositosta-5,7,10(19)-triene-1,3-diol.

Compound Ib is 1α-OH-22,23-dihydroporiferacalciferol. It can also benamed as(1S,3R)-(5Z,7E)-9,10-seco-22,23-dihydroporiferasta-5,7,10(19)-triene-1,3-diol.

Compound Ic is 1α-OH-stigma-calciferol. It can also be named as(1S,3R)-(5Z,7E,22E)-9,10-Secostigmasta-5,7,10(19),22-tetraene-1,3-diol.

Compound Id is 1α-OH-porifera-calciferol. It can also be named as(1S,3R)-(5Z,7E,22E)-9,10-Secoporiferasta-5,7,10(19),22-tetraene-1,3-diol.

Synthesis of CARD-024 is given for example in Moriarty et al. U.S. Pat.No. 6,900,191, the disclosure of which is hereby incorporated byreference. Other compounds of Formula (I) are made by analogoussynthetic routes.

According to any of these embodiments, the composition can bedeliverable, for example, by the oral route. Other routes ofadministration include without limitation parenteral, sublingual,buccal, intranasal, pulmonary, topical, transdermal, intradermal,ocular, otic, rectal, vaginal, intragastric, intracranial, intrasynovialand intra-articular routes. When the subject is able to swallow, it ispreferred to provide and administer the compositions in an oral form.These include without limitation tablets, capsules, gel caps, lozenges,and the like.

A composition prepared according to the invention comprises, in additionto the active ingredient selected from among compounds of Formula (I),one or more pharmaceutically acceptable excipients. The excipient orexcipients together form a pharmaceutically acceptable carrier.

If the composition is to be prepared in solid form for oraladministration, for example as a tablet or capsule, it typicallyincludes at least one or more solid diluents and one or more soliddisintegrants. Optionally, the excipients further include one or morebinding agents, wetting agents and/or antifrictional agents (lubricants,anti-adherents and/or glidants). Many excipients have two or morefunctions in a pharmaceutical composition. Characterization herein of aparticular excipient as having a certain function, e.g., diluent,disintegrant, binding agent, etc., should not be read as limiting tothat function. Depending on the desired dosing scheme, the composition,the carriers provide the dosage form with more or less instantaneousrelease, or are formulated to give a delayed release or a release spreadout over time. The latter are advantageous for patient compliance, as asubject need take fewer doses in a given time. For example, a full dailydose can be provided in an extended release formulation that need betaken only once a day. If desired, the compositions can be provided withenteric coatings that withstand the acid conditions of the stomach, butwhich release the active ingredient in the more basic confines of theduodenum or small intestine. Further information on excipients can befound in standard reference works such as Handbook of PharmaceuticalExcipients, 3rd ed. (Kibbe, ed. (2000), Washington: AmericanPharmaceutical Association).

Suitable diluents illustratively include, either individually or incombination, lactose, including anhydrous lactose and lactosemonohydrate; lactitol; maltitol; mannitol; sorbitol; xylitol; dextroseand dextrose monohydrate; fructose; sucrose and sucrose-based diluentssuch as compressible sugar, confectioner's sugar and sugar spheres;maltose; inositol; hydrolyzed cereal solids; starches (e.g., cornstarch, wheat starch, rice starch, potato starch, tapioca starch, etc.),starch components such as amylose and dextrates, and modified orprocessed starches such as pregelatinized starch; dextrins; cellulosesincluding powdered cellulose, microcrystalline cellulose, silicifiedmicrocrystalline cellulose, food grade sources of α- and amorphouscellulose and powdered cellulose, and cellulose acetate; calcium saltsincluding calcium carbonate, tribasic calcium phosphate, dibasic calciumphosphate dihydrate, monobasic calcium sulfate monohydrate, calciumsulfate and granular calcium lactate trihydrate; magnesium carbonate;magnesium oxide; bentonite; kaolin; sodium chloride; and the like. Suchdiluents, if present, typically constitute in total about 5% to about95%, for example about 20% to about 90%, or about 50% to about 85%, byweight of the composition. The diluent or diluents selected preferablyexhibit suitable flow properties and, where tablets are desired,compressibility.

Microcrystalline cellulose and silicified microcrystalline cellulose areparticularly useful diluents, and are optionally used in combinationwith a water-soluble diluent such as mannitol. Illustratively, asuitable weight ratio of microcrystalline cellulose or silicifiedmicrocrystalline cellulose to mannitol is about 10:1 to about 1:1, butratios outside this range can be useful in particular circumstances.

Suitable disintegrants include, either individually or in combination,starches including pregelatinized starch and sodium starch glycolate;clays; magnesium aluminum silicate; cellulose-based disintegrants suchas powdered cellulose, microcrystalline cellulose, methylcellulose,low-substituted hydroxypropylcellulose, carmellose, carmellose calcium,carmellose sodium and croscarmellose sodium; alginates; povidone;crospovidone; polacrilin potassium; gums such as agar, guar, locustbean, karaya, pectin and tragacanth gums; colloidal silicon dioxide; andthe like. One or more disintegrants, if present, typically constitute intotal about 0.2% to about 30%, for example about 0.5% to about 20%, orabout 1% to about 10%, by weight of the composition.

Binding agents or adhesives are useful excipients, particularly wherethe composition is in the form of a tablet. Such binding agents andadhesives should impart sufficient cohesion to the blend being tabletedto allow for normal processing operations such as sizing, lubrication,compression and packaging, but still allow the tablet to disintegrateand the composition to be absorbed upon ingestion. Suitable bindingagents and adhesives include, either individually or in combination,acacia; tragacanth; glucose; polydextrose; starch includingpregelatinized starch; gelatin; modified celluloses includingmethylcellulose, carmellose sodium, hydroxypropylmethylcellulose (HPMC),hydroxypropylcellulose, hydroxyethylcellulose and ethylcellulose;dextrins including maltodextrin; zein; alginic acid and salts of alginicacid, for example sodium alginate; magnesium aluminum silicate;bentonite; polyethylene glycol (PEG); polyethylene oxide; guar gum;polysaccharide acids; polyvinylpyrrolidone (povidone or PVP), forexample povidone K-15, K-30 and K-29/32; polyacrylic acids (carbomers);polymethacrylates; and the like. One or more binding agents and/oradhesives, if present, typically constitute in total about 0.5% to about25%, for example about 1% to about 15%, or about 1.5% to about 10%, byweight of the composition.

Povidone and hydroxypropylcellulose, either individually or incombination, are particularly useful binding agents for tabletformulations, and, if present, typically constitute about 0.5% to about15%, for example about 1% to about 10%, or about 2% to about 8%, byweight of the composition.

Wetting agents, if present, are normally selected to maintain the drugin close association with water, a condition that can improvebioavailability of the composition. Non-limiting examples of surfactantsthat can be used as wetting agents include, either individually or incombination, quaternary ammonium compounds, for example benzalkoniumchloride, benzethonium chloride and cetylpyridinium chloride; dioctylsodium sulfosuccinate; polyoxyethylene alkylphenyl ethers, for examplenonoxynol 9, nonoxynol 10 and octoxynol 9; poloxamers (polyoxyethyleneand polyoxypropylene block copolymers); polyoxyethylene fatty acidglycerides and oils, for example polyoxyethylene (8) caprylic/capricmono- and diglycerides, polyoxyethylene (35) castor oil andpolyoxyethylene (40) hydrogenated castor oil; polyoxyethylene alkylethers, for example ceteth-10, laureth-4, laureth-23, oleth-2, oleth-10,oleth-20, steareth-2, steareth-10, steareth-20, steareth-100 andpolyoxyethylene (20) cetostearyl ether; polyoxyethylene fatty acidesters, for example polyoxyethylene (20) stearate, polyoxyethylene (40)stearate and polyoxyethylene (100) stearate; sorbitan esters, forexample sorbitan monolaurate, sorbitan monooleate, sorbitanmonopalmitate and sorbitan monostearate; polyoxyethylene sorbitanesters, for example polysorbate 20 and polysorbate 80; propylene glycolfatty acid esters, for example propylene glycol laurate; sodium laurylsulfate; fatty acids and salts thereof, for example oleic acid, sodiumoleate and triethanolamine oleate; glyceryl fatty acid esters, forexample glyceryl monooleate, glyceryl monostearate and glycerylpalmitostearate; tyloxapol; and the like. One or more wetting agents, ifpresent, typically constitute in total about 0.1% to about 15%, forexample about 0.2% to about 10%, or about 0.5% to about 7%, by weight ofthe composition.

Nonionic surfactants, more particularly poloxamers, are examples ofwetting agents that can be useful herein. Illustratively, a poloxamersuch as Pluronic™ F127, if present, can constitute about 0.1% to about10%, for example about 0.2% to about 7%, or about 0.5% to about 5%, byweight of the composition.

Lubricants reduce friction between a tableting mixture and tabletingequipment during compression of tablet formulations. Suitable lubricantsinclude, either individually or in combination, glyceryl behenate;stearic acid and salts thereof, including magnesium, calcium and sodiumstearates; hydrogenated vegetable oils; glyceryl palmitostearate; talc;waxes; sodium benzoate; sodium acetate; sodium fumarate; sodium stearylfumarate; PEGs (e.g., PEG 4000 and PEG 6000); poloxamers; polyvinylalcohol; sodium oleate; sodium lauryl sulfate; magnesium lauryl sulfate;and the like. One or more lubricants, if present, typically constitutein total about 0.05% to about 10%, for example about 0.1% to about 5%,or about 0.2% to about 2%, by weight of the composition. Sodium stearylfumarate is a particularly useful lubricant.

Anti-adherents reduce sticking of a tablet formulation to equipmentsurfaces. Suitable anti-adherents include, either individually or incombination, talc, colloidal silicon dioxide, starch, DL-leucine, sodiumlauryl sulfate and metallic stearates. One or more anti-adherents, ifpresent, typically constitute in total about 0.05% to about 10%, forexample about 0.1% to about 7%, or about 0.2% to about 5%, by weight ofthe composition. Colloidal silicon dioxide is a particularly usefulanti-adherent.

Glidants improve flow properties and reduce static in a tabletingmixture. Suitable glidants include, either individually or incombination, colloidal silicon dioxide, starch, powdered cellulose,sodium lauryl sulfate, magnesium trisilicate and metallic stearates. Oneor more glidants, if present, typically constitute in total about 0.05%to about 10%, for example about 0.1% to about 7%, or about 0.2% to about5%, by weight of the composition. Colloidal silicon dioxide is aparticularly useful glidant.

Other excipients such as buffering agents, stabilizers, antioxidants,antimicrobials, colorants, flavors and sweeteners are known in thepharmaceutical art and can be used in compositions of the presentinvention. Tablets can be uncoated or can comprise a core that iscoated, for example with a nonfunctional film or a release-modifying orenteric coating. Capsules can have hard or soft shells comprising, forexample, gelatin (in the form of hard gelatin capsules or soft elasticgelatin capsules), starch, carrageenan and/or HPMC, optionally togetherwith one or more plasticizers.

A solid orally deliverable composition of the present invention is notlimited by any process used to prepare it. Any suitable process ofpharmacy can be used, including dry blending with or without directcompression, and wet or dry granulation.

If the composition is to be prepared in liquid (including encapsulatedliquid) form, the active ingredient (a compound according to Formula I)can be, for example, dissolved in a suitable carrier, typically onecomprising a lipid solvent for the API. The higher the unit dose, themore desirable it becomes to select a carrier that permits a relativelyhigh concentration of the drug in solution therein. In variousembodiments, the concentration of active ingredient in the carrier is atleast about 10 mcg/ml (mcg stands for micrograms), e.g., about 10 toabout 500 mcg/ml, but lower and higher concentrations can be acceptableor achievable in specific cases. Illustratively, the drug concentrationin various embodiments is at least about 10 mcg/ml, e.g., about 10 toabout 250 mcg/ml, or at least about 20 mcg/ml, e.g., about 20 to about200 mcg/ml, for example about 20, about 25, about 30, about 40, about50, about 75, about 100 or about 150 mcg/ml.

The subject can be human or non-human (e.g., a farm, zoo, work orcompanion animal, or a laboratory animal used as a model) but in animportant embodiment the subject is a human patient in need of the drug,for example to treat a disease characterized by fibrosis, especially ofthe bowel as in inflammatory bowel disease or Crohn's disease. A humansubject can be male or female and of any age, but is typically an adult.

The composition is normally administered in an amount providing atherapeutically effective daily dose of the drug. The term “daily dose”herein means the amount of drug administered per day, regardless of thefrequency of administration. For example, if the subject receives a unitdose of 150 mcg twice daily, the daily dose is 300 mcg. Use of the term“daily dose” will be understood not to imply that the specified dosageamount is necessarily administered once daily. However, in a particularembodiment the dosing frequency is once daily (q.d.), and the daily doseand unit dose are in this embodiment the same thing.

What constitutes a therapeutically effective dose depends on thebioavailability of the particular formulation, the subject (includingspecies and body weight of the subject), the disease to be treated, thestage and/or severity of the disease, the individual subject's toleranceof the compound, whether the compound is administered in monotherapy orin combination with one or more other drugs, and other factors. Thus,the daily dose can vary within wide margins, for example from about 10to about 1000 mcg. Based on body weight, the human effective dose isexpected to be on the order of 0.2 to 5 mcg per kilogram of body weight(0.2-5 mcg/kg), for example in the range of 0.5-2 mcg/kg. In anon-limiting illustration, the effective dose is on the order of 25-150mcg per subject, assuming body weights of 50-150 kilograms. Greater orlesser daily doses can be appropriate in specific situations, and thephysician will vary the dosage and titrate the effective amountaccording to best medical judgment. It will be understood thatrecitation herein of a “therapeutically effective” dose herein does notnecessarily require that the drug be therapeutically effective if only asingle such dose is administered; typically therapeutic efficacy dependson the composition being administered repeatedly according to a regimeninvolving appropriate frequency and duration of administration. It isstrongly preferred that, while the daily dose selected is sufficient toprovide benefit in terms of treating bowel disease, it should not besufficient to provoke an adverse side-effect to an unacceptable orintolerable degree. A suitable therapeutically effective dose can beselected by the physician of ordinary skill without undueexperimentation based on the disclosure herein and on art cited herein,taking into account factors such as those mentioned above. The physicianmay, for example, start a patient on a course of therapy with arelatively low daily dose and titrate the dose upwards over a period ofdays or weeks, to reduce risk of adverse side-effects.

Illustratively, suitable doses of CARD-024 are generally about 10 toabout 500 mcg, about 10 to about 250 mcg, about 25 to about 400 mcg,about 25 to about 200 mcg, for example about 5, about 10, about 20,about 25, about 50, about 75, about 100, about 150, about 175, about200, about 250, about 500, about 750 or about 1000 mcg, administered asneeded at an average dosage interval of about 3 hours to about 7 days,for example about 8 hours to about 3 days, or about 12 hours to about 2days. In most cases a once-daily (q.d.) administration regimen issuitable.

An “average dosage interval” herein is defined as a span of time, forexample one day or one week, divided by the number of unit dosesadministered over that span of time. For example, where a drug isadministered three times a day, around 8 am, around noon and around 6pm, the average dosage interval is 8 hours (a 24-hour time span dividedby 3). If the drug is formulated as a discrete dosage form such as atablet or capsule, a plurality (e.g., 2 to about 10) of dosage formsadministered at one time is considered a unit dose for the purpose ofdefining the average dosage interval.

If desired, one or more agents typically used to treat inflammatorybowel disease may be used in a combination therapy along with a compoundor mixture of compounds of Formula I in the methods and compositions ofthe invention. In various embodiments, methods of treating IBD and CDinclude administering a composition containing one or more compounds ofFormula I and further containing additional active agents. Non-limitingexamples of such agents include biologics (e.g., inflixamab, adelimumab,and CDP-870), an immunomodulatory compound such as interleukin-10,interleukin-4, or a TNFα inhibitor, small molecule immunomodulators(e.g., VX 702, SCIO 469, doramapimod, RO 30201195, SCIO 323, DPC 333,pranalcasan, mycophenolate, and merimepodib), non-steroidalimmunophilin-dependent immunosuppressants (e.g., cyclosporine,tacrolimus, pimecrolimus, and ISAtx247), 5-amino salicylic acid,mesalamine, sulfasalazine, balsalazide disodium, and olsalazine sodium,methotrexate, azathioprine, and alosetron. Thus, in one embodiment, theinvention involves the combination of a compound of Formula I and any ofthe foregoing agents, and methods of treating inflammatory bowel diseaseor Crohn's disease by administering them to a subject.

Discussion

Administering compositions comprising compounds of Formula (I) has beendemonstrated to be effective at relieving fibrosis and treating thesymptoms of inflammatory bowel disease such as, without limitation,Crohn's disease.

Crohn's disease (CD) is characterized by cycles of intestinalinflammation (flares) and mucosal healing. While potentanti-inflammatory therapies reduce inflammation and disease symptoms,the need for anti-fibrotic therapies to prevent the inexorabledevelopment of fibrostenotic disease remains (Szabo et al., 2010).Intestinal fibrosis remains a significant serious complication of CD,often culminating in surgical intervention (Van Assche et al., 2010). Inaddition, patients with CD frequently have vitamin D deficiency, thoughwhether a cause or a consequence of intestinal malabsorption remainsopen to debate (Harris et al., 2008; Siffledeen et al., 2003). Innumerous other organ systems, vitamin D deficiency is associated withfibrosis. Treatment with vitamin D analogs reduces fibrosis both in cellculture and in animal models of fibrosis, suggesting that treatment withvitamin D analogs could reduce intestinal fibrosis (Li et al., 2005;Mancuso et al., 2008; Tan et al., 2006; Weishaar et al., 1990; Zhang etal., 2010).

In the intestine, subepithelial myofibroblasts contribute to intestinalwound healing in part by reconstituting the ECM while aberrantmyofibroblast activation is postulated to produce fibrosis (Powell etal., 1999; Pucilowska et al., 2000). In CD, activated or dysregulatedintestinal myofibroblasts are the major source of excessive ECM andsubsequent fibrosis (Powell et al., 1999).

As seen in primary intestinal myofibroblasts (Brenmoehl et al., 2009;Simmons et al., 2002), TGFβ stimulation of human colonic myofibroblastCcd-18co cells induced a pro-fibrotic phenotype, characterized byincreased αSMA protein expression, actin stress fibers, and mature focaladhesions. As disclosed herein, co-treatment with CARD-024, a compoundwith little hypercalcemic effect, reduced αSMA stimulation by TGFβ in adose-dependent manner. CARD-024 treatment repressed the gene expressionof endothelin-1 which is involved in myofibroblast differentiation andfibrosis (Guidry and Hook, 1991).

In the matrix stiffness model, human colonic myofibroblasts demonstratedmorphological changes when cultured on collagen-coated polyacrylamidegels of increasing matrix stiffness. CARD-024 treatment attenuatedmorphological changes induced by high matrix substrates, including theorganization of actin stress fibers, development of mature focaladhesions, and elongated dendritic processes. Actin stress fiberassembly is regulated by MLCK (Anderson et al., 2004). In colonicmyofibroblasts, MLCK was transcriptionally induced by high matrixstiffness but repressed by CARD-024, suggesting that the morphologicaleffects of CARD-024 occur through interruption of MLCK-mediated actinassembly.

Activated myofibroblasts are characterized by the expression of aSMA(Powell et al., 1999). Recent work in pulmonary, hepatic, and dermalmyofibroblasts demonstrated that a stiff extracellular matrix induces apro-fibrotic, activated phenotype characterized by increased αSMAexpression (Jones and Ehrlich, 2011; Liu et al., 2010; Olsen et al.,2011). In colonic myofibroblasts, high matrix stiffness induced αSMAprotein expression while treatment with CARD-024 blocked induction ofαSMA protein expression.

Myofibroblast differentiation is (in part) dependent on FAK (focaladhesion kinase) signaling (Brenmoehl et al., 2009). FAK is a criticalmediator of cytoskeletal responses including proliferation, migration,and adhesion. (Schaller, 2010) In addition, FAK mediates mechanosensingof the extracellular environment via an integrin signaling pathway(Assoian and Klein, 2008; Parsons, 2003). In colonic myofibroblasts,high matrix stiffness induced pFAK while CARD-024 repressed FAKactivation suggesting that CARD-024 may inhibit pFAK signaling.Endothelin-1 (ET-1), a soluble fibrogenic peptide, has been shown tophosphorylate FAK in fibroblasts (Daher et al., 2008; Kennedy et al.,2008) while vitamin D antagonizes ET-1 stimulation in cardiac myocytes(Wu et al., 1996). In our stiffness model, ET-1 transcription is inducedby high matrix stiffness and repressed by CARD-024, suggesting thatvitamin D analogs may target the FAK/ET-1 pathway. However, CARD-024treatment did not repress the expression of the classic fibrogenic genescol1A1 and Fn-1. Collagen expression is regulated in part by both extra-and intracellular calcium levels (Fitzgerald et al., 2006; Nakade etal., 2001). CARD-024 has minimal effects on calcium metabolism,therefore failure to repress collagen gene expression may due to thecalcium-mediated regulation of collagen gene expression Vitamin D andits metabolites have been reported to repress or induce fibronectinexpression, depending on the cell type (Ezzat and Asa, 2005; Ramirez etal., 2010). Fibronectin expression is necessary for cell adhesion andattenuation of fibronectin expression may reduce cellular responsivenessto vitamin D or its analogs (Ezzat and Asa, 2005).

Recently, the COX-2/PGE2 pathway has been described as mediating thepro-fibrotic response to matrix stiffness (Liu et al., 2010). Though ourwork did not conclusively demonstrate that high matrix stiffnessrepresses COX-2/PTGS2 in colonic myofibroblasts, a repressive trend wasobserved in high matrix stiffness while CARD-024 treatment showed astimulatory trend in both low and high matrix stiffness's. While themechanism of matrix stiffness regulation of COX-2 is unknown, COX-2expression in intestinal myofibroblasts is regulated by IL-1β(Hinterleitner et al., 1996). In colonic myofibroblasts cultured on lowor high stiffness matrices, IL-1β is transcriptionally repressed by highstiffness. In macrophages, vitamin D (1,25-Dihydroxyvitamin D3(1,25(OH)2D3), stimulates IL-1β in macrophages. (Lee et al., 2011)CARD-024 markedly induced IL-1β expression irrespective of matrixstiffness, suggesting CARD-024 may target an IL-1β/COX-2 pathway.

Intestinal fibrosis and vitamin D deficiency are two frequentcomplications of Crohn's disease. The high proportion of CD patientsultimately requiring surgical intervention underscores the need foreffective anti-fibrotic medical therapies. This study demonstrates thatCARD-024, a compound with minimal hypercalcemic effects, reduces thefibrogenic response of intestinal myofibroblasts.

These results support monitoring and maintaining normal vitamin D levelsin Crohn's disease patients with a history of fibrostenotic disease.

EXAMPLES

Human recombinant TGFβ1 was obtained from R&D Systems (Minneapolis,Minn.). CARD-024 (1-alpha-hydroxyvitamin D5) was provided by R. Simpson(Cardiavent Inc., Ann Arbor, Mich.) and dissolved in 100% ethanol. Allchemicals were purchased from Sigma (Sigma-Aldrich, Saint Louis, Mo.),except where noted.

Cell Culture

Colonic human myofibroblast Ccd-18co cells (CRL-1459 from ATCC) werecultured in alpha-MEM (Invitrogen, Carlsbad, Calif.) supplemented with10% fetal bovine serum and sub-cultured weekly.

Cells were plated at 30-40% confluence. To stimulate a fibroticphenotype, Ccd-18co cells were serum-starved for 24 h prior to treatmentwith 1 ng/ml TGFβ or 1 ng/ml TGFβ and increasing doses of CARD-024(10-1000 nM) for 48 h. Given ethanol was used to dilute CARD-024,ethanol was added to the untreated and TGFβ treated cells to a finalconcentration of 0.1%. For stiffness experiments, low-passage numberCcd-18co cells were seeded at 1×105 cells/ml on 6-well plates containingcollagen-coated acrylamide gels corresponding to soft (4.3 kPa, 0.02%bisacrylamide) or stiff (28.1 kPa, 0.16%) matrices. Cells were allowedto attach to the matrix for 4 h and then the gels were transferred tonew wells to avoid paracrine signaling from cells attached to theplastic well bottom. For the CARD-024 stiffness experiments, cells wereplated as described above and serum-starved overnight prior to treatmentwith 1000 nM of CARD-024 diluted in 100% ethanol for 24 h. The 1000 nMCARD-024 dose was selected from the results of the TGFβ experiments.Since ethanol was used to dilute CARD-024, ethanol was added tountreated cells to a final concentration of 0.1%. Experiments wereperformed on early (4 to 7) passage cells. Cultures were routinelyassayed for mycoplasma contamination.

Matrix Stiffness Gels

Collagen-coated polyacrylamide gels corresponding to physiologicalstiffnesses of 2.6 kPa to 28.1 kPa were generated using varying ratiosof 40% acrylamide to 2% bisacrylamide (Bio-Rad, Hercules, Calif.) (Aplinand Hughes, 1981; Pelham and Wang, 1998). Specifically, an aqueoussolution of 0.10% ammonium persulfate (Bio-Rad), 0.15% TEMED (Bio-Rad),40% acrylamide solution was supplemented with 0.01%, 0.02%, 0.08%, or0.16% bisacrylamide. The acrylamide gels were polymerized on a NaOHtreated, amino-silanated (3-aminopropyltriethoxysilane), andglutaraldehyde treated 25 mm round glass coverslip. 10 ml of eachacrylamide/bisacrylamide solution was filter sterilized and pipettedunto a chloro-silanated glass surface treated withdichlorodimethylsilane (DCDMS) and a treated coverslip was inverted ontothe acrylamide/biacrylamide solution. The acrylamide matrix was allowedto polymerize for 10 min between the two surfaces and transferred to a6-well tissue culture plate containing sterile PBS. As determined by aseries of stability experiments (data not shown), the acrylamide gelswere stable for at least 1 month. For all experiments, gels were usedwithin a month.

Prior to seeding with colonic myofibroblasts, the acrylamide gels werecollagen-coated. 0.2 mg/ml of sulfo-SANPAH (Thermo Scientific, Rockford,Ill.) was added to each well containing the acrylamide gel-coated coverslip and UV crosslinked at a wavelength of 254 nm in a Stratagene UVcrosslinker oven (Stratagene, La Jolla, Calif.) at a 5-inch distancefrom the UV source for 10 min. The gels were coated with 0.2 mg/ml ofrat tail collagen I (BD Biosciences, Bedford, Mass.) overnight at 37° C.with gentle agitation. Excess collagen was removed by several washeswith sterile PBS. Gels were UV sterilized for 30 min prior to seedingwith colonic myofibroblasts. Cells were serum-starved overnight prior totreatment with CARD-024. From the results of the TGFβ treatmentexperiments, 1000 nM CARD-024 was selected for all stiffness matrixexperiments.

Control cells received ethanol at the same final concentration (0.1%) tocontrol for the ethanol concentration in CARD-024 solution.

Microelastometer Measurements

Microelastometer measurements were determined foracrylamide/bisacrylamide gels ranging from 2.6 to 28.1 kPa bisacrylamideusing a microelastometer (Micro-Elastometer, Artann Laboratories, WestTrenton, N.J.) (Egorov et al., 2008). For the microelastometermeasurements, gels were synthesized without a NaOH-treated glasscoverslip.

For measurements, thicker gels were used than for cell plating sinceprevious experiments determined a larger material height (substratethickness) was needed to generate accurate and reproduciblemicroelastomer measurements (data not shown).

By measuring vertical displacement of the gels, the stress (force perunit area) and strain (material compression in response to force) weredetermined by the microelastometer. Raw stress and strain values wereplotted and the slope of the stress-strain curve was determined usingthe region between the first and second points of inflection tocalculate the Young's Modulus (kPa), an expression of substratestiffness.

Protein Isolation and Western Blotting

Immunoblotting was utilized for the detection of α-smooth muscle actin.Ccd-18Co cells were washed in ice-cold PBS containing proteaseinhibitors (Roche, Indianapolis, Ind.), then lysed in ice-cold RIPAbuffer (1% Igepal CA 630, 1% sodium deoxycholate, 0.1% SDS, 0.15 M NaCl,2 mM EDTA, 50 mM NaF) with a 1:100 dilution of protease inhibitorcocktail III (Calbiochem, La Jolla, Calif.) and 2 mM sodiumorthovanadate.

Total protein was separated by SDS polyacrylamide gel electrophoresis(Bio-Rad) with prestained protein markers loaded for molecular massdetermination (Invitrogen, Carlsbad, Calif.), and transferred to PVDFmembranes (Amersham Biosciences, Piscataway, N.J.). Membranes wereblocked in 5% milk/TBST solution for 1 h at room temperature orovernight at 4° C. α-smooth muscle actin was detected by incubating themembrane overnight at 4° C. with mouse anti-human monoclonal antibody(Sigma, St. Louis, Mo.) at 1:5000 dilution in 5% milk/TBST.

pFAK was detected using a rabbit polyclonal phosphospecific antibodyagainst Tyr-397-FAK (Invitrogen) at 1:5000. As a loading control, amouse antibody for GAPDH (Chemicon, Temecula, Calif.) was used. Afterwashing, the membranes were incubated with the appropriate secondaryantibody (anti-mouse IgG+HRP or anti-rabbit IgG+HRP, Amersham,Piscataway, N.J.) for 1 h at room temperature and the signal wasdetected by the Pierce detection system (Pierce, Rockford, Ill.).Autoradiographs were scanned and quantitated using ImageJ analysissoftware (NIH, Bethesda, Md.).

Microscopy

Cells were photographed using a Leica (Leica Microsystems Inc., BuffaloGrove, Ill.) DMIRB inverted microscope and photographed with an OlympusDP-30 camera (Center Valley, Pa.). Cell length was determined fromphotomicrographs using ImageJ (NIH, Bethesda, Md.). Cell count wasdetermined from a minimum of 3 representative photographs of a 100×magnification field. 92 L. A. Johnson et al./Experimental and MolecularPathology 93 (2012) 91-98.

Immunofluorescence

Expression of activated myofibroblast markers was analyzed by confocalimmunofluorescence microscopy using the Olympus FluoView™ FV500/IXsystem (Olympus America, Center Valley, Pa.) at the University ofMichigan Microscopy and Image Analysis Laboratory. Ccd-18Co cells wereseeded onto collagen-coated polyacrylamide gels (0.02% bis or 0.16% bis)attached to glass coverslips. After 24 h, gels were rinsed with PBS,then were fixed with 4% paraformaldehyde (Electron Microscopy Sciences,Hatfield, Pa.) in PBS for 5 min, followed by permeabilization with 0.2%Triton X-100 (LabChem, Pittsburgh, Pa.) for 15 min. Gels were rinsed 3times with PBS, then were pre-blocked with SFX signal enhancer(Invitrogen, Carlsbad, Calif.), and blocked with 20% goat serum(Invitrogen, Carlsbad, Calif.).

For visualization of focal adhesions, the gels were incubated for 2 h atroom temperature with mouse anti-vinculin primary antibody (Sigma, St.Louis, Mo.) at 1:250 in PBS/0.1% Triton X-100. The gels were rinsedthree times with PBS/0.1% Triton-X100, followed by incubation with Alexa555-conjugated goat anti-mouse secondary antibody (Molecular Probes,Eugene, Oreg.) for 30 min, at room temperature, in PBS/0.1% TritonX-100. Gels were washed 6 times, followed by incubation with phalloidin(Sigma, St. Louis, Mo.) at 1:100, overnight, at 4° C., for visualizationof actin stress fibers. Cells were co-stained with4,6′-diamidino-2-phenylindole (DAPI), (Molecular Probes, Eugene, Oreg.)to visualize nuclei. The gels were mounted with ProLong Gold mountingmedium (Invitrogen, Carlsbad, Calif.) prior to imaging by confocalimmunofluorescence microscopy.

Quantitative RT-PCR

RNA from Ccd-18co cells was extracted using the RNeasy kit (Qiagen,Valencia, Calif.). cDNA was generated by reverse transcription of 1 μgof total RNA using the Superscript First Strand RT kit (Invitrogen,Carlsbad, Calif.). Quantitative real-time PCR (qPCR) was performed forMLCK, IL-1β, PTGS2, ET-1, and GAPDH with the TaqMan gene expressionassays (ABI, Foster City, Calif.) on a Bio-Rad iCycler real-time PCRsystem. Cycling conditions were 95° C. 10 min, followed by 40 cycles of95° C. 15 s and 62° C. 60 s. Gene expression was normalized to GAPDH asthe endogenous control, and fold-changes (RQ) relative to untreatedcontrols were calculated using the ΔΔCt-method (Livak and Schmittgen,2001).

Statistical Analysis

Comparisons between multiple were analyzed with ANOVA, while pairwisecomparisons of two groups were performed with Student's t test.

Results

Example 1 Dose Response of Colonic Myofibroblasts to CARD-024

To induce a pro-fibrotic phenotype, human colonic myofibroblasts(Ccd-18co cells) were treated with increasing doses of TGFβ1 (0.1-2ng/ml). Consistent with (Simmons et al., 2002), TGFβ induced apro-fibrotic response as characterized by a dose-dependent increase inαSMA protein expression (FIG. 1A) and increased actin stress fiber andfocal adhesion staining (data not shown).

To determine whether CARD-024 represses fibrogenesis in colonicmyofibroblasts, Ccd-18co cells were stimulated with TGFβ and treatedwith 10-1000 nM CARD-024 (FIG. 1B). In colonic myofibroblasts stimulatedwith TGFβ, increasing doses of CARD-024 attenuated αSMA proteinexpression in a dose-responsive manner. At the highest dose (1000 nM)CARD-024 attenuated αSMA protein expression 2-fold (p=0.012) (FIG. 1C).

Example 2 Development of Collagen-Coated Polyacrylamide Gels

Typical culture of lung myofibroblasts on rigid plastic substrates hasprofound effects on myofibroblast differentiation and activation (Hinz,2010). In lung myofibroblasts, matrix stiffness induces changes in cellphenotype and function from a quiescent, non-proliferative phenotype toan activated, ECM-secreting phenotype (Hinz, 2010).

Given that CARD-024 repressed αSMA protein expression on the rigidplastic matrix, we postulated that CARD-024 might have an anti-fibroticeffect on colonic myofibroblasts cultured on a more physiologicallycompliant substrate.

To determine the effect of matrix stiffness on colonic myofibroblastphenotype, collagen-coated polyacrylamide gels were generated asdetailed in the Experimental Procedures. Varying the ratio of acrylamideto bisacrylamide from 0.01% to 0.16% produced substrates withstiffnesses from 2.6 to 28.1 kPa as determined by microelastometermeasurements.

Similar to published work in myofibroblasts from other tissues, Ccd-18cocolonic myofibroblasts cultured on these matrices demonstrated a gradedmonotonic change in morphology with increasing matrix stiffness. On lowstiffness substrates (2.6 and 4.3 kPa), cells exhibited anundifferentiated phenotype, characterized by a rounded appearance, witha few cells displaying small dendritic processes. However the dendriticprocesses were markedly truncated and less numerous compared to thetypical myofibroblast morphology on the plastic substrate (FIG. 2). Incontrast, cells cultured on the high stiffness substrates (15.6 and 28.1kPa) exhibited a more differentiated morphology, with a characteristicstellate appearance and multiple, elongated dendritic processesresembling myofibroblasts cultured on plastic. Compared to cellscultured on the low matrix stiffness, myofibroblasts cultured on thehigh stiffness substrates had more actin stress fiber staining withmature focal adhesions apparent in the highest matrix stiffness (datanot shown).

Example 3 Effects of CARD-024 and Substrate Stiffness on MyofibroblastMorphology

To determine whether CARD-024 altered myofibroblast morphology, weanalyzed the effect of CARD-024 treatment on colonic myofibroblastscultured on low (4.3 kPa) and high (28.1 kPa) stiffness substrates. Forthe low stiffness substrate, 4.3 kPa gels were selected as the 2.6 kPagels proved difficult to image by confocal microscopy.

Ccd-18co cells cultured on the high stiffness (28.1 kPa) matrixdeveloped a stellate morphology, with multiple dendritic processescompared to myofibroblasts grown on the low stiffness (4.3 kPa) matrix(FIG. 3B). Similar to human foreskin fibroblasts, which developelongated dendritic processes in response to increase matrix stiffness(Jones and Ehrlich, 2011), colonic myofibroblasts on the stiff substratehad significantly elongated dendritic processes compared tomyofibroblasts cultured on the soft substrate (173 μm vs 133 μm,p=0.004)(FIG. 3C). High matrix stiffness also increased the number ofcells>2.5-fold compared to the soft matrix (66.5 vs 25 cells/200× field,p=0.0043) (FIG. 3D).

Treatment with CARD-024 repressed the effects of matrix stiffness oncell morphology, with cell morphology on the high stiffness substratesin the presence of CARD-024 that was quite similar to the myofibroblastson low stiffness substrates (FIG. 3B). Dendrite length was nearlyidentical (146 vs 145 μm, p=0.95) between CARD-024 treatedmyofibroblasts on low compared to high stiffness matrices (FIG. 3C).While treatment with CARD-024 did not significantly decrease the numberof myofibroblasts on the stiff substrate, a 50% reduction in cell numberwas observed compared to untreated cells on the stiff matrix, suggestinga non-significant trend toward decreased cell number with CARD-024treatment.

Activated myofibroblasts are characterized by the development of actinstress fibers and focal adhesions (Hinz, 2010). Colonic myofibroblastscultured on the soft substrate had fewer actin stress fibers and diffusevinculin staining without organized focal adhesions (FIG. 3D). Incontrast, myofibroblasts cultured on the stiff matrix exhibited anactivated morphology with increased numbers of actin stress fibers andmature, well-defined focal adhesions (FIG. 3D). On the stiff substrate,treatment with CARD-024 produced poorly organized immature focaladhesions with diffuse cytoplasmic staining, suggesting CARD-024inhibited focal adhesion maturation (FIG. 3D).

Example 4 Effect of CARD-024 and Substrate Stiffness on ECM Protein andGene Expression

Similar to the pro-fibrotic effects of TGFβ stimulation, high matrixstiffness induced αSMA protein expression in colonic myofibroblasts(FIG. 4A). Treatment with CARD-024 repressed αSMA expression on the highstiffness substrate to levels indistinguishable from the low stiffnesssubstrate. Myofibroblast differentiation and cytoskeletal reorganizationis dependent upon a number of factors, including FAK (focal adhesionkinase) signaling (Brenmoehl et al., 2009). In colonic myofibroblasts,the high stiffness matrix induced phosphorylation of focal adhesionkinase (FAK), while CARD-024 inhibited FAK phosphorylation, suggestingCARD-024 affects pFAK signaling (FIG. 4B).

Actin stress fiber formation is regulated in part bymyosin light chainkinase (MLCK) (Anderson et al., 2004). Given that CARD-024 repressed thedevelopment of actin stress fibers and mature focal adhesions, weexamined the role of matrix stiffness and CARD-024 upon MLCK geneexpression. MLCK was induced 2-fold (p=0.004) in myofibroblasts on thehigh compared to a low stiffness substrate (FIG. 4C). Treatment withCARD-024 significantly repressed MLCK expression to levels comparable tountreated cells on the low stiffness substrate (FIG. 4C).

Similarly, the high stiffness substrate significantly induced ET-1 geneexpression by 60% (p=0.009) (FIG. 4D). Treatment with CARD-024significantly repressed ET-1 expression in high stiffness conditions tolevels indistinguishable from low stiffness conditions.

In pulmonary myofibroblasts, matrix stiffness suppresses an endogenousCOX-2/PGE2 inhibitory pathway (Liu et al., 2010). While increased matrixstiffness attenuated but did not significantly repress PTGS2 (whichencodes the COX-2 enzyme), treatment with CARD-024 induced PTGS2 at bothlow and high stiffness, suggesting CARD-024 may affect the COX-2/PGE2pathway (FIG. 4E). In intestinal myofibroblasts, IL-1β stimulates COX-2expression (Hinterleitner et al., 1996). Therefore we examined theeffect of matrix stiffness and CARD-024 on IL-1β expression.

High matrix stiffness attenuated IL-1β expression, though this did notachieve statistical significance (p=0.069). Treatment with CARD-024significantly induced IL-1β expression above low stiffness levels with a4-fold induction at low stiffness (pb0.0001) and 1.8-fold induction athigh stiffness (p=0.002) (FIG. 4F).

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We claim:
 1. A method of treating a subject to relieve the symptoms ofinflammatory bowel disease, the method comprising administering to thesubject a therapeutically effective dose of a compound of Formula (I)

wherein Et is ethyl, wherein the dashed line indicates a single bondbetween carbon 22 and carbon 23, and wherein the configuration at carbon24 to which Et is attached is in the R configuration or the Sconfiguration.
 2. The method according to claim 1, wherein the compoundof Formula (I) comprises(1S,3R)-(5Z,7E)-9,10-secositosta-5,7,10(19)-triene-1,3-diol (CARD-024).3. The method according to claim 1, comprising administering acomposition comprising a compound of Formula (I) and a pharmaceuticallyacceptable carrier.
 4. The method according to claim 3, wherein thecomposition comprises a tablet.
 5. The method according to claim 1,wherein the subject is diagnosed with Crohn's disease.
 6. The methodaccording to claim 2, comprising administering the effective doseorally.
 7. The method of claim 2, wherein the daily dose is from 10 mcgto 1000 mcg.
 8. The method of claim 2, further comprising administeringto the patient a compound selected from infliximab, adelimumab,certolizumab (CDP-870), interleukin-10,interleukin-4,6-[(Aminocarbonyl)(2,6-difluorophenyl)amino]-2-(2,4-difluorophenyl)-3-pyridinecarboxamide(VX 702),6-Chloro-5-[[(2R,5S)-4-[(4-flurophenyl)methyl]-2,5-dimethyl-1-piperazinyl]carbonyl]-N,N,1-trimethyl-α-oxo-1H-Indole-3-acetamide (SCIO 469), doramapimod,((2R)-2-((3R)-3-amino-3{4-[2-methyl-4-quinolinyl)methoxy]phenyl}-2-oxopyrrolidinyl)-N-hydroxy-4-methylpentanamide)) (DPC333), pranalcasan, mycophenolate, merimepodib, cyclosporine, tacrolimus,pimecrolimus,6[(2S,3R,4R,6E)-3-hydroxy-4-methyl-2-(methylamino)-6,8-nonadienoicacid]-Cyclosporin A (ISAtx247), 5-aminosalicylic acid, mesalamine,sulfasalazine, balsalazide disodium, olsalazine sodium, methotrexate,azathioprine, and alosetron.
 9. The method of claim 2, comprisingadministering to the patient a composition comprising CARD-024 and acompound selected from infliximab, adelimumab, certolizumab (CDP-870),interleukin-10,interleukin-4,6-[(Aminocarbonyl)(2,6-difluorophenyl)amino]-2-(2,4-difluorophenyl)-3-pyridinecarboxamide(VX 702),6-Chloro-5-[[(2R,5S)-4-[(4-flurophenyl)methyl]-2,5-dimethyl-1-piperazinyl]carbonyl]-N,N,1-trimethyl-α-oxo-1H-Indole-3-acetamide (SCIO 469), doramapimod,((2R)-2-((3R)-3-amino-3{4-[2-methyl-4-quinolinyl)methoxy]phenyl}-2-oxopyrrolidinyl)-N-hydroxy-4-methylpentanamide)) (DPC333), pranalcasan, mycophenolate, merimepodib, cyclosporine, tacrolimus,pimecrolimus,6-[(2S,3R,4R,6E)-3-hydroxy-4-methyl-2-(methylamino)-6,8-nonadienoicacid]-Cyclosporin A (ISAtx247), 5-aminosalicylic acid, mesalamine,sulfasalazine, balsalazide disodium, olsalazine sodium, methotrexate,azathioprine, and alosetron.